Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
2000-05-25
2003-05-20
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S278000, C800S288000, C435S069100, C435S069800, C435S101000, C435S419000, C435S468000, C536S023700
Reexamination Certificate
active
06566585
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to transgenic plant cells and plants with an increased activity of an amylosucrase protein and an increased activity of a branching enzyme. Such plant cells and plants synthesize a modified starch and/or synthesize &agr;-1,6 branched &agr;-1,4-glucans with a modified branching degree in O-6-position and/or give a higher yield in comparison with corresponding genetically non-modified wild type plants (plant cells).
In the area of agriculture and forestry it has been a permanent endeavor to produce plants with increased yield, in particular, in order to ensure the food for the continuously growing population of the world and to guarantee the supply of regenerating raw materials. Traditionally, attempts have been made to obtain productive plants by breeding. For each plant species of interest a corresponding breeding program has to be carried out. This is, however, time- and work-intensive. Progress has been made, partly by genetic manipulation of plants, i.e. by purposeful introduction and expression of recombinant nucleic acid molecules in plants. Such approaches have the advantage that, in general, they are not being limited to one plant species but can be transferred to other plant species. Therefore it seems desirable to provide plant cells and plants which give increased yields as well as to offer methods for the production of such plant cells and plants.
With regard to the growing importance which has been attached to vegetable substances as a source of regenerating raw material recently, it is one of the tasks in biotechnological research to strive towards adjusting these vegetable raw materials to the demands of the manufacturing industry. In order to facilitate the use of regenerating raw materials in as many application areas as possible it is furthermore essential to achieve a great variety of substances. Moreover, it is necessary to increase the yield of these vegetable substances in order to increase the efficiency of the production of sources of regenerating vegetable raw materials.
Apart from oils, fats and proteins, polysaccharides are the most important regenerating vegetable raw materials. Apart from cellulose, starch plays a vital role with the polysaccharides as it is one of the most important reserve substance in higher plants.
Apart from its use in foods, the polysaccharide starch is also widely used as regenerating raw material for the production of industrial products. The polysaccharide starch is composed of chemically uniform basic components, the glucose molecules, but forms a complex mixture of various molecules which have differing polymerization and branching degrees and therefore differ substantially in their physical and chemical properties.
A differentiation is made between amylose starch, a basically non-branched polymer composed of (&agr;-1,4-glycosidically linked glucose units, and the amylopectin starch, a branched polymer wherein branching is caused by the occurrence of additional &agr;-1,6-glycosidic links. According to the literature (Voet and Voet, Biochemistry, John Wiley & Sons, 1990) &agr;-1,6-glycosidic links occur on average at every 24
th
to every 30
th
glucose residue. This corresponds to a branching degree of about 3%-4%. Details of the branching degree are variable and depend on the source (e.g. plant species, plant variety, etc.) of the individual starch. Plants typically used for the industrial production of starch vary in their amylose content of the total starch content between 10 and 25%.
In order to facilitate a very wide use of polysaccharides such as e.g. starch it seems desirable to provide plants which are modified in their polysaccharide composition and, for example, are able to synthesize modified starch and/or highly branched &agr;-1,6-&agr;-1,4-glucans which are particularly suitable for various uses. One possibility to produce such plants is—apart form breeding methods—the purposeful modification of the starch metabolism in starch producing plants by genetic engineering methods, A prerequisite hereto, however, is the identification and characterization of the enzymes playing a role in the starch synthesis and/or modification as well as the isolation of the corresponding DNA molecules encoding these enzymes. The biochemical synthesis pathways which lead to the formation of starch are essentially known. The starch synthesis in plant cells takes place in the plastids. In photosynthetically active tissues these are the chloroplasts, in photosynthetically inactive starch-storing tissues the amyloplasts.
The most important enzymes participating in the starch synthesis are the starch synthases (cf. for example patent application WO 96/15248), the R1-enzyme (cf. for example WO 97/11188) as well as the branching enzymes (cf. for example WO 92/14827). The exact role of other enzymes such as e.g. the starch phosphorylases (cf. for example WO 98/40503) during starch biosynthesis is not known. In order to provide further possibilities to modify any plants in such a way that they synthesize modified starch, it is also possible to introduce foreign nucleic acid molecules, as e.g. bacterial or fungal, which are not present in wild type plants and which encode proteins participating in the synthesis of polysaccharides. It could be shown, for example, that the synthesis of so-called “Amylofructan” is possible by amyloplastidic expression of bacterial fructosyltransferases in amyloplasts (Smeekens, Trends in Plant Science Vol. 2 No. 8 (1997), 286-288).
The heterologous expression of a bacterial glycogen synthase in potato plants leads to a slight decrease in the amylose content, an increase of the branching degree and a change in the branching pattern of the amylopectin in comparison with wild type plants (Shewmaker et al., Plant. Physiol., 104 (1994), 1159-1166).
Moreover, the expression of a bacterial branching enzyme in potato plants in amylose-free potato mutants (amf) (Jacobsen et al., Euphytica, 44 (1989), 43-48) leads to amylopectin molecules having 25% more branching points (Kortstee et al., The Plant Journal, 10(1), (1996), 83-90) than the control molecules (amf). The increase in branching points was due to a modification of the distribution of the chain length of longer side chains in favor of shorter side chains. The reduction of the average chain-length and the reduction of the &lgr;max after iodine staining also are an indication for a higher branched structure of the amylopectin in transformed plants in comparison with non-transformed plants (Kortstee et al., see above). The branching degree of glycogen of about 10% could, however, not be achieved via this approach. Glycogen, a polysaccharide, which is found mainly in animals and bacteria, contains highly-branched &agr;-1,6-&agr;-1,4-glucans. Glycogen differs from starch also in the average length of the side chains and in the polymerization degree. According to the literature (Voet and Voet, Biochemistry, John Wiley & Sons, 1990) it contains an &agr;-1,6-branching point at every 8
th
to 12th glucose residue on average. This corresponds to a branching degree of about 8% to 12%. There are various figures for the molecular weight of glycogen which vary between 1 million and more than 1000 millions (D. J. Manners in: Advances in Carbohydrate Chemistry, Ed. M. L. Wolfrom, Academic Press, New York (1957), 261-298; Geddes et al., Carbohydr. Res., 261(1994), 79-89). Theses figures, too, very much depend on the corresponding source organism, its nutritional state as well as the kind of isolation of glycogen. Usually it is obtained by costly and time-intensive methods from mussels (e.g. Mytillus edulis), from mammal livers or muscles (e.g. rabbits, rats) (Bell et al., Biochem. J. 28 (1934), 882; Bueding and Orrell, J. Biol. Chem., 236 (1961), 2854). Moreover, in plants one finds, for example, in the su1-mutant of maize the so-called phytoglycogen which has a branching degree of about 10% and which shows, in comparison with amylopectin a modified side chain distribution (Yun and Matheson, Carbohydrate Research 243, (1993),
Fox David T.
PlantTec Biotechnologie GmbH
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